Biomedical subjects

Aharon G Freud

Publications and source records attributed to Aharon G Freud.

10 recordsLinked to original sources

PubMed2026

Identification of elements determining KIR gene demethylation at the CD56-bright stage of NK cell development.

The variegated expression of the KIR family of class I MHC receptors generates specialized natural killer (NK) cells capable of allele-specific HLA recognition. Understanding the mechanism of KIR gene activation will lead to improved methods for the generation of fully functional NK cells. A central RUNX-binding site in the KIR proximal promoter is required for gene activation. RUNX proteins recruit ten-eleven translocation (TET) proteins that generate 5-hydroxymethylcytosine (5hmC) and drive DNA demethylation. Assessment of 5-methylcytosine (5mC) and 5hmC residues at four stages of NK cell development reveals deposition of 5hmC primarily in a CREB site next to the RUNX site at the CD56Bright stage but not the subsequent CD56Dim stage representing fully mature NK cells. KIR promoter demethylation is delayed relative to other lineage-associated genes, indicating a high threshold for KIR gene demethylation in developing NK cells, and a window of opportunity for RUNX/TET-dependent KIR gene activation in CD56Bright NK cells.

6-base sequencing↗

PubMed2006

Hlx homeobox transcription factor negatively regulates interferon-gamma production in monokine-activated natural killer cells.

Natural killer (NK) cells contribute to host immunity, including tumor surveillance, through the production of interferon gamma (IFN-gamma). Although there is some knowledge about molecular mechanisms that induce IFN-gamma in NK cells, considerably less is known about the mechanisms that reduce its expression. Here, we investigate the role of the Hlx transcription factor in IFN-gamma production by NK cells. Hlx expression is induced in monokine-activated NK cells, but with delayed kinetics compared to IFN-gamma. Ectopic Hlx expression decreases IFN-gamma synthesis in primary human NK cells and IFN-gamma promoter activity in an NK-like cell line. Hlx protein levels inversely correlate with those of STAT4, a requisite factor for optimal IFN-gamma transcription. Mechanistically, we provide evidence indicating that Hlx overexpression accelerates dephosphorylation and proteasome-dependent degradation of the active Y693-phosphorylated form of STAT4. Thus, Hlx expression in activated NK cells temporally controls and limits the monokine-induced production of IFN-gamma, in part through the targeted depletion of STAT4.

Animals↗

PubMed2006

A phase I study of ultra low dose interleukin-2 and stem cell factor in patients with HIV infection or HIV and cancer.

PURPOSE: Ultra low doses of interleukin-2 (IL-2) can activate the high-affinity IL-2 receptor constitutively expressed on CD56(bright) natural killer (NK) cells, the CD34+ NK cell precursor, and CD4+ CD25+ regulatory T cells (Tregs) in vivo. We have previously shown synergy between IL-2 and stem cell factor (SCF) in the generation of CD56(bright) NK cells from CD34+ hemopoietic progenitor cells in vitro and showed synergistic NK cell expansion in an in vivo preclinical model. To determine the safety, toxicity, and immune modulation of this combination of cytokines in vivo, we conducted a first-in-man phase I study. EXPERIMENTAL DESIGN: A phase I dose escalation study was conducted using IL-2 at 900,000 or 650,000 IU/m2/d for 8 weeks with 5 or 10 microg/kg/d of SCF given thrice a week for 8 weeks in patients with HIV infection and/or cancer. RESULTS: Ten of 13 patients completed therapy; four experienced the dose-limiting toxicities of grade 3 fatigue or urticaria. The maximum tolerated doses of IL-2 and SCF in combination is 650,000 IU/m2/d of IL-2 and 5 microg/kg/d thrice a week of SCF. NK cells were expanded over 2-fold on therapy; Tregs were expanded nearly 6-fold from baseline. CONCLUSIONS: Administration of IL-2 with SCF is safe and well tolerated and leads to expansion of lymphocyte subsets in patients with HIV or HIV and cancer; however, the changes in NK cell and Treg expansion seen with this cytokine combination were no different than those seen with a similar dose of IL-2 alone.

Adult↗

PubMed2006

Evidence for discrete stages of human natural killer cell differentiation in vivo.

Human natural killer (NK) cells originate from CD34(+) hematopoietic progenitor cells, but the discrete stages of NK cell differentiation in vivo have not been elucidated. We identify and functionally characterize, from human lymph nodes and tonsils, four NK cell developmental intermediates spanning the continuum of differentiation from a CD34(+) NK cell progenitor to a functionally mature NK cell. Analyses of each intermediate stage for CD34, CD117, and CD94 cell surface expression, lineage differentiation potentials, capacity for cytokine production and natural cytotoxicity, and ETS-1, GATA-3, and T-BET expression provide evidence for a new model of human NK cell differentiation in secondary lymphoid tissues.

Biomarkers↗

PubMed2006

Human natural killer cell development.

Our understanding of human natural killer (NK) cell development lags far behind that of human B- or T-cell development. Much of our recent knowledge of this incomplete picture comes from experimental animal models that have aided in identifying fundamental in vivo processes, including those controlling NK cell homeostasis, self-tolerance, and the generation of a diverse NK cell repertoire. However, it has been difficult to fully understand the mechanistic details of NK cell development in humans, primarily because the in vivo cellular intermediates and microenvironments of this developmental pathway have remained elusive. Although there is general consensus that NK cell development occurs primarily within the bone marrow (BM), recent data implicate secondary lymphoid tissues as principal sites of NK cell development in humans. The strongest evidence stems from the observation that the newly described stages of human NK cell development are naturally and selectively enriched within lymph nodes and tonsils compared with blood and BM. In the current review, we provide an overview of these recent findings and discuss these in the context of existing tenets in the field of lymphocyte development.

Cell Differentiation↗

PubMed2005

IL-15 but not IL-2 rapidly induces lethal xenogeneic graft-versus-host disease.

Interleukin-2 (IL-2) and IL-15 are structurally related cytokines that share receptor components but display markedly different effects in multiple in vivo model systems. Here we demonstrate that IL-15 but not IL-2 exacerbates xenogeneic graft-versus-host disease (X-GVHD) in severe combined immunodeficient murine recipients of human peripheral-blood lymphocytes (hu-PBL-SCID). Treatment of hu-PBL-SCID mice with IL-15 resulted in rapid fatality, lymphocytic infiltrations in the liver, lung, and spleen consistent with X-GVHD, and a marked expansion of human CD4+ and CD8+ T cells compared with controls. Depletion of human T cells in vivo abrogated the lethality of IL-15 treatment. To our knowledge, these data are the first to demonstrate in vivo activation and expansion of human T lymphocytes in response to IL-15 with concomitant exacerbation of human T-cell-mediated X-GVHD.

Adjuvants, Immunologic↗

PubMed2005

A human CD34(+) subset resides in lymph nodes and differentiates into CD56bright natural killer cells.

In humans, T cells differentiate in thymus and B cells develop in bone marrow (BM), but the natural killer (NK) precursor cell(s) and site(s) of NK development are unclear. The CD56bright NK subset predominates in lymph nodes (LN) and produces abundant cytokines compared to the cytolytic CD56dim NK cell that predominates in blood. Here, we identify a novel CD34dimCD45RA(+) hematopoietic precursor cell (HPC) that is integrin alpha4beta7bright. CD34dimCD45RA(+)beta7bright HPCs constitute <1% of BM CD34(+) HPCs and approximately 6% of blood CD34(+) HPCs, but >95% of LN CD34(+) HPCs. They reside in the parafollicular T cell regions of LN with CD56bright NK cells, and when stimulated by IL-15, IL-2, or activated LN T cells, they become CD56bright NK cells. The data identify a new NK precursor and support a model of human NK development in which BM-derived CD34dimCD45RA(+)beta7bright HPCs reside in LN where endogenous cytokines drive their differentiation to CD56bright NK cells in vivo.

Antigens, CD34↗

PubMed2004

Failed adoptive immunotherapy with tumor-specific T cells: reversal with low-dose interleukin 15 but not low-dose interleukin 2.

Adoptive immunotherapy with tumor-specific T cells has emerged as a valid approach for prevention or treatment of diseases, such as melanoma and EBV-associated lymphoma. As interleukin (IL) 15 promotes survival of CD8(+) memory CTLs, we hypothesized that it could be used to enhance antitumor immunity in vivo through the maintenance of adoptively transferred memory CTL. To test this, we treated mice bearing P1A(+) tumors with adoptively transferred T cells possessing a transgenic Valpha8(+) T-cell receptor specific for the P1A tumor antigen (called P1CTL). Mice were then randomized to receive daily low-dose IL-15 (0.5 microg/day) or PBS. Mice receiving the transgenic P1CTL and IL-15 experienced a significantly delayed tumor relapse or complete tumor regression (P < 0.002 compared with PBS), with a striking persistence of the CD8(+) Valpha8(+) P1CTL compared with mice receiving the CD8(+) Valpha8(+) P1CTL and PBS vehicle (26.3 versus 5.1% P < 10(-5)). Animals exhibiting complete tumor regression had a significant population of CD8(+) Valpha8(+) P1CTL (46%) that persisted with IL-15 treatment until 140 days after adoptive transfer and successfully defended them against tumor rechallenge without IL-15. Low-dose IL-2 afforded no protection over vehicle and resulted in lower percentages of T cells with an activated memory phenotype, lower Bcl-2 expression, and lower ex vivo antitumor cytotoxicity compared with mice treated with IL-15. Collectively, the data support the notion that exogenous low-dose IL-15 therapy can enhance and even reverse the limited efficacy of adoptively transferred tumor-specific T-cell therapy and may do so in a fashion that is superior and distinct from exogenous IL-2 therapy.

Animals↗

PubMed2004

Selective efficacy of depsipeptide in a xenograft model of Epstein-Barr virus-positive lymphoproliferative disorder.

BACKGROUND: Immune-compromised individuals are at increased risk for developing aggressive Epstein-Barr virus (EBV)-associated lymphoproliferative disorders after primary EBV infection or for reactivation of a preexisting latent EBV infection. We evaluated the effect of depsipeptide, a histone deacetylase inhibitor, on EBV-positive lymphoblastoid cell lines (LCLs) and Burkitt lymphoma cell lines in a mouse model and explored its mechanism of action in vitro. METHODS: We studied EBV-transformed LCLs, which express a latent III (Lat-III) viral gene profile, as do some EBV-positive lymphoproliferative malignancies, and Burkitt lymphoma cell lines, which express a Lat-I viral gene profile. Cell lines were used to characterize depsipeptide-induced apoptosis, which was evaluated by flow cytometry. Flow cytometry, western blot analyses, and histone deacetylase inhibitors were used to investigate components of prodeath and survival pathways in vitro. We studied depsipeptide's effects on survival with a mouse xenograft model of EBV-positive human B-cell tumors (groups of 10 mice). All statistical tests were two-sided. RESULTS: Depsipeptide (5 mg/m2 of body surface area) treatment was associated with statistically significantly improved survival of mice carrying Lat-III EBV-positive LCL tumors, compared with that of control-treated mice (day 30: for depsipeptide-treated mice, 90% survival, 95% confidence interval [CI] = 73.2% to 100%; for control-treated mice, 20% survival, 95% CI = 5.79% to 69.1%; P<.001), but it was not associated with survival of mice carrying Lat-I EBV-positive Burkitt lymphoma tumors. Depsipeptide induced apoptosis in 64% of LCLs and in 14% of EBV-positive Burkitt lymphoma cells in vitro. Depsipeptide-treated LCL cultures had two distinct cell populations--one sensitive and one resistant to depsipeptide. Depsipeptide-mediated apoptosis was associated with a 12-fold increased level of active caspase 3, but some apoptosis persisted despite z-VAD-fmk treatment to inhibit caspase activity. Depsipeptide-resistant LCLs expressed higher levels of latent membrane protein 1 (LMP1; P = .017), BCL2 (P = .032), and nuclear factor kappaB (NF-kappaB) (P<.001) than depsipeptide-sensitive LCLs; this resistance was circumvented by treatment with PS-1145, an inhibitor of NF-kappaB activation (P<.001). CONCLUSIONS: Apoptosis is induced by depsipeptide via caspase-dependent and -independent pathways in Lat-III EBV-positive LCLs and is enhanced by inhibiting NF-kappaB activity. Depsipeptide as a treatment for Lat-III EBV-associated lymphoproliferative disorders should be explored further in clinical trials.

Animals↗

PubMed2003

Safety of interleukin-12 gene therapy against cancer: a murine biodistribution and toxicity study.

As a prerequisite for a human clinical trial using interleukin (IL)-12 gene therapy, the biodistribution and safety of IL-12, administered as an intradermal naked DNA injection, was evaluated in mice. The pNGVL3-mIL12 plasmid used in this study is a nonviral vector designed to induce a high level of IL-12 protein expression during a transient transfection of the host cell. The biodistribution was evaluated by a polymerase chain reaction (PCR) assay that is capable of detecting less than 100 copies of the plasmid in the context of host DNA. Twenty-four hours after three intradermal injections of 0.5 microg or 5 microg of pNGVL3-mIL12 plasmid, the plasmid was detectable in various internal organs, the blood, and the injection site. The plasmid was detectable in the gonads of only one animal at the high-dose treatment 24 hr after the injections. In the majority of the organs the plasmid was undetectable throughout the study. Possible side effects were monitored by histology and clinical chemistry, and the level of IL-12 protein expression was assessed by enzyme-linked immunosorbent assay (ELISA). No treatment-related histologic abnormalities were detected and the blood chemistry parameters showed no toxicity. The IL-12 protein was undetectable at all times at the injection site and interferon (IFN)-gamma levels at the injection site and in the serum were at background levels. The results of this murine safety study indicate that based on the distribution pattern of the plasmid in the body and the undetectable toxicities in the tissues, the use of the pNGVL3-hIL12 plasmid in cancer gene therapy clinical trials can be considered as safe.

Animals↗